A molecular investigation of Friend SFFV is proposed. When infected into mice, this virus induces rapid polyclonal erythroid hyperplasia, followed by outgrowth of a monoclonal erythroleukemia. The SFFV genome encodes a deleted version of an MCF-like glycoprotein, gp55, which functions as an oncogene for this virus. Previous investigators have shown that SFFV infection confers erythropoietin independence to infected erythroid progenitors. While he was a graduate student and postdoctoral, Dr. Li did seminal work on this system.In particular, he showed that gp55 can physically bind to the erythropoietin receptor. Moreover, this binding is responsible for induction of erythropoietin-independent growth of Ba/F3-ER cells. He also generalized this finding, to show that Friend and Moloney MCF gp70s can induce erythropoietin and IL-2 independent growth of Ba/F3-ER and Ba/F3-I2 cells respectively. In the current proposal, Dr. Li proposes to further investigate the mechanism of gp55- induced erythropoietin independence. There are three experimental aims. 1) The necessity of gp55-EpoR interactions occurring at the cells surface (suggested in Dr. Li's previous experiments) will be tested. This will be accomplished by generating MAbs to gp55 and EpoR, and testing if they block SFFV leukemogenesis. Other experiments will test gp55 mutants defective in cell surface expression, or glycosulation for leukemogenesis. The role of the TM domain in gp55 will also be investigated. The effect of inhibiting EpoR export to the cell surface will also be tested. 2) Variants of Friend and Moloney MCFs grown in Ba/F-ER or Ba/F-I2 cells arise that have gp55-like deletions in the env region. Continued passage of one of them (M2) gave rise to a highly pathogenic virus M2v. The critical regions of these variants will be identified by molecular cloning and introduction into retroviral vectors, followed by sequencing.3) The mechanism of signal transduction after gp55 binds EpoR will be investigated. First, the effect of gp55 expression on EpoR turnover will be measured. Second, subtractive cloning will be used to identify genes that are up-regulated in epo-independent gp55- expressing cells.